Investigation of Campylobacter fetus in breeding bulls of private farms in Bangladesh

Abstract Background Bovine genital campylobacteriosis (BGC) is a venereal disease caused by Campylobacter fetus that has a negative impact on animal reproduction. The bull is considered to be a symptomless carrier that spreads the disease agent to breeding cows, causing infertility and sporadic abortion. Aim The study aims to estimate the prevalence, identify risk factors of Campylobacter fetus (C. fetus) infection and antimicrobial resistance pattern of the C. fetus isolates. Method A cross‐sectional survey was conducted in Mymensingh district of Bangladesh. Bull smegma samples (single sample from each bull) were collected from 300 bulls from four farms and tested via culture, biochemical identification and finally 16S rRNA and cdtA gene‐based molecular assays (PCR) for herd and animal‐level prevalence estimation. Herd‐ and animal‐level data on risk factors were collected from the farmers using a pretested questionnaire and analysed by univariable and multivariable logistic regression models with a p value of <0.05 was taken statistically significant for both analyses. Results Among the surveyed farms, 75% (95% CI: 19.4%–99.4%) were confirmed to have bulls infected with Campylobacter fetus at herd level. However, animal‐level occurrence of C. fetus was estimated to be 8.7% (26/300) (95% CI: 5.7%–12.4%). Natural service increases the odds of campylobacteriosis 38.18 times (95% CI: 13.89–104.94) in comparison to artificial insemination for C. fetus infection in bulls. Significantly, half of the isolates (50%, n = 13) were identified to be multidrug resistant (MDR) for three to five antimicrobial agents. Conclusion This study highlights the need to develop official guidelines for C. fetus control and prevention in Bangladesh including mandatory artificial insemination in reproductive cows and heifers, routine screening of breeding bulls for C. fetus free status.


INTRODUCTION
of the aborted fetus are used to confirm the presence of C. fetus infection in cattle herds (Campero et al., 2005;Garcia et al., 1983;Penner, 1988). Interestingly, infection in breeding bulls does not hamper sperm quality or even libido (Eaglesome & Garcia, 1992;Van Bergen et al., 2005). Therefore, bulls should be considered using a targeted approach for herd health screening and for epidemiological studies to build strategies for prevention and control.
Bovine genital campylobacteriosis is transmitted primarily through natural mating in cattle breeding program; however, infection is also spread via infected bull semen used in artificial insemination (AI) and/contaminated equipment (Modolo et al., 2000). Transmitted between female cattle is less likely since the disease usually spreads through natural mating/coitus (Clark, 1971;Hoffer, 1981); however, transmission between bulls may occur during mounting when distance between breeding bulls is inadequate, and several bulls are kept in the same house (Taylor, 2002). Infection with BCG does not hamper conception; however, it usually culminates with early embryonic loss, and therefore, delayed oestrus, while abortions in the infected female are most frequently detected between 4 and 6 months of pregnancy period. The disease is generally self-limiting in females, and the acquired immunity usually persists for several years (Mshelia et al., 2007;Taylor, 2002). The bulls can also be exposed to Campylobacter fetus infection from the infected cows during natural mating. The pathogens grow in the crypts of the penis and preputial sac; however, they are not able to invade into the deeper tissue mass. A bull may either be infected temporarily or over a period year. Therefore, the bulls can act as a reservoir and disseminate this pathogen through coitus (Eaglesome & Garcia, 1992). Nevertheless, long-lasting infec-tions are observed in aged bulls due to presence of deeper penile and preputial crypts where the organisms reside. Bulls, which contract infection from diseased cows during breeding, do not develop convalescent immunity, and therefore, mostly remain susceptible to reinfection.
Growing demand for animal origin food especially red meat is estimated to be increased twofold by 2050 in small and middle income countries (Agus & Widi, 2018).To mitigate such demand numerous initiatives have been taken by the government through intervention of many projects and programs for the development of livestock sectors since several decades ago. These activities have directed to improve the productivity of native cattle for genetic improvements by artificial insemination (Department of Livestock Sevices, 2007).
There are approximately 24.39 million cattle, 1.49 million buffaloes, 26.4 million goats, and 3.6 million sheep in Bangladesh in 2019(Department of Livestock Sevices, 2020. At present, among the total cattle population, 15% are high yielding crossbred stocks (Hamid et al., 2017) of Holstein Friesian, Sindhi, Sahiwal with a small proportion of Jersey breed (Miazi et al., 2007. As the majority of the female cattle in Bangladesh are not taken under the artificial insemination program which may increase the likelihood of transmission C. fetus infection in cows through natural mating from the infected bulls (Clark, 1971;Hoffer, 1981 This is the first study in Bangladesh to assess the presence of C.
fetus infection in breeding bulls through molecular detection and risk factor assessment that will assist in formulating strategies to prevent and control measures for reproductive health promotion of cattle.

Study design and location
A cross-sectional survey was conducted during January 2019 to June 2020 in Mymensingh district, a promising cattle rearing zone of Bangladesh with an estimated 923,000 heads of cattle (Department of Livestock Sevices, 2017), of which 15% are crossbred farmed cattle (Hamid et al., 2017).

F I G U R E 1
Location of study farms in Mymensingh district of Bangladesh, 4 breeding farms were included under this study, of which 3 farms (1 indigenous and 2 crossbred bull farms) from Mymensingh Sadar subdistrict and 1 bull farm from Bhaluka subdistrict of Mymensingh district were included under this study. for the production of frozen semen in private and government breeding farms. So, we think this sample will represent the breeding bull populations in Bangladesh ( Figure 1).

Data collection
The data and samples were collected by an expert veterinarian and one veterinary paraprofessional. A semi-structured questionnaire was used for collection data from farmers/farm managers during data collection and subsequent animal sampling (Table S1). The questionnaire was designed to capture variables related to (i) herd-level determinants (10 questions) and (ii) animal-level determinants (7 questions).
Apart from field interview of the herd managers, some herd-level data were also obtained and/or validated through a transect walking method (like breed, husbandry type, presence of biogas plant, biosecurity status, etc.). The responses of the questionnaire were coded and stored in Excel data sheet for descriptive and inferential analyses.

Sampling procedure
Since bulls are usually asymptomatic carriers of C. fetus associated with BGC infections in the endemic herd (BonDurant, 2005), samples were collected from the preputial crypts of the penis of the bulls. The smegma samples were obtained by scraping from the preputial and penile mucosa using a disposable plastic scraper in 50 mL falcon tube (Tedesco et al., 1977), afterwards, rinsed in 20-30 mL of phosphate buffered saline (PBS) following method established previously (Mordasini et al., 2006).
Aseptic procedures were maintained during the collection of the samples, which were transported, maintaining a cold chain at 4-6 • C, to the laboratory and processed within 4-6 h of collection. A schematic F I G U R E 2 Schematic diagram of sample collection and evaluation workflow.
diagram of sample collection and testing workflow is presented in

Culture-based methods and biochemical tests
Samples were subjected to a filtration method using cellulose filter with a pore size 0.65 µm (Biotech, Göttingen, Germany) as described previously (Bolton et al., 1988), with minor adjustment. Briefly, 100 µL of each sample was spread on the filters that were kept on the surface of blood agar base (no. 2) (HiMedia, Mumbai, India) with supplementation of 5% sheep blood and growth supplement (HiMedia, Mumbai, India) to isolate C. fetus (World Organization for Animal Health, 2021), and then allowed to stand at room temperature for 30 min.
Afterwards, the filters were removed and the media were incubated at 37 • C for 48 h under microaerophilic conditions, that is, 5-10% oxygen, 5-10% carbon dioxide and 5-9% hydrogen for optimal growth of the campylobacters (Vandamme, 2000), using AnaeroPouch ® -MicroAero (Mitsubishi Gas Chemical Co., Inc., Tokyo, Japan). Colonies having the typical morphological traits of Campylobacter spp., (i.e., grey, flat and irregularly spreading colonies) grown on the incubated media were selected. The Gram staining was performed on the selected colonies and the Gram-negative curved-shaped cells, observed under a light microscope, were considered as presumptive Campylobacter spp. A single and pure colony was obtained after subculture of the presumptive Campylobacter colonies using blood agar media. A tentative confirmation of the isolates was achieved based on growth characteristics and biochemical tests, including catalase test, oxidase test, hippurate hydrolysis test, nitrate reduction test, indoxyl acetate test and 1% glycerine test as per standard protocols (Foster et al., 2004;Nachamkin, 2003;Swai et al., 2005).

2.5.2
Polymerase chain reaction (PCR) The tentative Campylobacter isolates were subjected to a genusspecific PCR assay to validate the presence of Campylobacter spp. A pure culture of tentative Campylobacter spp. was used for DNA extraction via boiling method (Hoshino et al., 1998). The amplification of the 16SrRNA gene using oligonucleotide primers was done following previously established method (Samosornsuk et al., 2007) as shown in the Table 1. Finally, a multiplex PCR assay targeting cdtA gene was done as serial testing for molecular identification of different species of Campylobacter (i.e., C. jejuni, C. coli and C. fetus) according to the method of Asakura et al. (2008) (Table 1). However, after confirmation by this multiplex PCR assay, the isolates were further screened using hipO genebased PCR to validate the identification of C. jejuni (Linton et al., 1997) ( Table 1 and Figure S1).
In the multiplex PCR assay, DNA templates of C. jejuni ATCC 33560, C. coli ATCC 33559 and C. fetus ATCC 27374 strains were used as positive controls, whereas Escherichia coli ATCC 25922 strain was used as a negative control. PCR products were subjected to a gel electrophoresis (1.5% agarose, Invitrogen, Carlsbad, CA, USA), and subsequently, stained with ethidium bromide (0.5 g/mL) and de-stained with distilled water, for 10 min each. Subsequently, the gel images were captured using a UV transilluminator (Biometra, Göttingen, Germany).

Antimicrobial susceptibility testing
All of the isolated strains of C. fetus were checked for antimicrobial susceptibility using the disk diffusion method (Luangtongkum et al., 2007)

Statistical analysis
Data generated through field interview and laboratory testing were Data on demographic and risk factors were summarised using descriptive statistics. In this evaluation, mean and standard deviation (SD) were estimated for continuous variables; however, proportions and frequency distributions were calculated for categorical variables.
All continuous variables like age of the animal, body weight were categorised before inclusion for logistic regression analysis.

Univariable mixed-effect logistic regression analyses
Univariable mixed effect logistic regression analyses were performed by including herd as a random intercepts (Bates et al., 2016). In this evaluation, C. fetus infection status as the response and each risk indicators in turn as an explanatory variable in this model. Collinearity among the variables was evaluated by Cramer's phi-prime statistic (R package 'vcd' , 'assocstats' function (Meyer et al., 2017). When Cramer's phiprime statistic was >0.70, a pair of variables was considered collinear . The herd-level data were not suitable for inferential interpretation since the number of herds were very small (n = 4).

Descriptive statistics
The survey was conducted on a sample group of 300 bulls from Mymensingh district of Bangladesh ( Figure 1). Among the four bull farms, the median number of bulls in each herd was 43 (12-203).
Of the total population of bulls, 96% (n = 288) were high-yielding crossbred variety, comprising mostly of Holstein Frisian crossbred, and some were Sahiwal/Jersey crossbred. All the farms were established for at least 5 years. Three of the farms under intensive farming with good biosecurity standards. In two of the study farms, feeding of bulls included the ready-made commercial feed (TMR). Half (n = 2, 50%) of the study farms practiced commercial artificial insemination program (

Herd-and animal-level prevalence
Among the four farms, three were found to be positive ( (Table 3). However, this study additionally confirmed the occurrence of C. jejuni in 11 samples, an apparent prevalence of 3.7% (95% CI: 1.8-6.5%) of the bulls smegma samples ( Figure 2).

Evaluation of animal-level risk factors
Seven variables were included in the univariable mixed-effects logistic regression analysis. Among the variables, breeding program was found to be significantly associated with animal-level C. fetus infectionpositive status. Natural service increases the odds of campylobacteriosis 38.18 times (95% CI: 13.89-104.94) in comparison to artificial insemination in breeding bulls (Table 4). Since a single variable was found to be significant univariable mixed-effects logistic regression analyses; therefore, building multivariable mixed-effect logistic regression model was not possible.

Antimicrobial resistance status
Among the 26 strains of C. fetus, only 7.7% (n = 2) demonstrated full resistance against a single antimicrobial agent, AMX or ERY or AZM,   (Table 5).

DISCUSSION
In this study, we evaluated animal-level occurrence, molecular traits and antimicrobial resistance (AMR) status of C. fetus isolated from four breeding bull farms of Mymensingh district in Bangladesh. The study further estimated potential risk factors for the occurrence of C. fetus in individual breeding bulls. The study confirmed the occurrence of C.
fetus in 75% (3/4) farms with an estimated animal/sample-level prevalence of 8.7% (26/300). Natural service was significantly associated with C. fetus positive status of breeding bulls in these selected farms.
The study recommends that annual screening of C. fetus in the breeding bulls can lead to reduced transmission in reproductive female cattle for a profitable dairy farming in Bangladesh.
This is the study for the first study of C. fetus of breeding bulls in Bangladesh. Since there is no published report on the occurrence of C.
fetus in bull samples in Bangladesh, the study findings provide an initial estimate and baseline. The apparent prevalence of C. fetus occurrence in bull samples in this study is comparable with results from other countries. The bacterial prevalence was reported to be 8%-26% in samples collected from Brazil in cows, fetuses and bulls (de Oliveira Filho et al., 2018;Stynen et al., 2003;Ziech et al., 2014), 16.4% of bulls in Nigeria (Mai et al., 2013), 10%-15% of cows in Malawi (Klastrup & Halliwell, 1977), and 6% in India from cattle faecal samples (Mshelia et al., 2010).
A lower-level prevalence of C. fetus, ranging 1%-5% of samples from bulls and cows, was reported for some herds in Brazil, Argentina and Canada (Campero et al., 2017;Devenish et al., 2005;de Oliveira et al., 2015;Molina et al., 2013). However, a higher-level prevalence of C.
fetus was also documented in cattle in Brazil (Miranda, 2005;Pellegrin et al., 2002;Rocha et al., 2009). A study conducted in India confirmed a high prevalence of C. fetus positive status as 47⋅6%, 3⋅3% and 25% from of vaginal swabs, preputial wash and cervicovaginal mucous samples, respectively collected from cows and bulls (Ishtifaq et al., 2020). The Previous investigations have claimed that exotic breeds (Bos taurus) are 4.2 times more likely to be susceptible to BGC, than indigenous cattle (Mai et al., 2013). However, our study has observed a significantly higher prevalence of C. fetus in bull herds comprising indigenous cattle (100% positive herd) than exotic breeds. The indigenous cattle in Bangladesh are frequently being subjected to natural breeding program, which may bring more likelihood of C. fetus-positive status through exposure of infection from the infected cows during coitus.
Natural breeding scheme is considered as the main contributing factor for the maintenance of C. fetus in the herd (BonDurant, 2005). Use of natural breeding program rather than artificial insemination was found to be important risk factor BGC in the present study. Nevertheless, C. fetus was detected for one of the two larger farms in our study ( Figure 2, Farm A and D) known to be practicing commercial artificial insemination (AI) breeding program with annual screening for C. fetus in the bull herds. Frequent infection through artificial insemination can happen in the territories where microbiological screening specifically targeting C. fetus is not well addressed or strictly adhered to (Irons et al., 2004;World Organization for Animal Health, 2018). Additionally, history of venereal disease in animal and mounting behaviour (mounting to other animals) would be potential risk factors of C. fetus occurrence in bulls, also noted (Taylor, 2002); however, these were found to be insignificant in this study.
Increasing incidence of antimicrobial resistance in Campylobacter spp. is a global concern. The World Health Organization ( Bangladesh, C. jejuni and C. coli being isolated from farmed animals, predominantly poultry, have been reported by a number of studies Islam et al., 2018;Kabir et al., 2014;Neogi et al., 2020).
However, systematic investigations observing antimicrobial resistance in C. fetus strains isolated from bull herds have so far received little focus. In this regard, data obtained in the present study can be considered as useful base-line information. Resistance to tetracycline, amoxicillin and streptomycin among a significant portion of C. fetus strains obtained from the bull smegma specimens was 6.2%, 34.6% and 34.6%, respectively, is a concern. Moreover, nearly half of the C. fetus strains (46.1%) were resistant to ciprofloxacin, and nearly one-third (34.6%) of these strains were also found to be resistant to gentamicin. Similar higher occurrence of resistance to these antimicrobial agents has been also reported in C. jejuni and C. coli in poultry from Bangladesh Islam et al., 2018;Kabir et al., 2014;Neogi et al., 2020).
A study in Canada revealed that C. fetus isolates in faecal samples had substantial resistance to tetracycline, doxycycline and ciprofloxacin (Inglis et al., 2006 (Garcia, 2009;Lage & Leite, 2000). The findings of this study support evidence-based decision-making to prioritise annual/seasonal screening of breeding bulls for C. fetus contamination to confirm breeding soundness. Infected bulls should be segregated from herds; however, a culling option requires a compensatory financing mechanism where resource constraints currently exist.
This is also an inherent limitation of a cross-sectional study design since exposure and outcome variables are collected simultaneously. However, more information on additional risk factors could be obtained from case-control (outcome is recorded first and the frequency of exposure recorded) or cohort studies (exposure is recorded first and followed for the outcome). Moreover, multi-locus sequence typing, differentiation into subspecies and phylogenetic analysis to estimate clonal relatedness would be required for a comprehensive understanding of the diversity, dynamics and source tracking of C. fetus populations circulating in the cattle herds. Therefore, future studies with in-depth molecular analysis to estimate genomic diversity in C.
fetus strains to be obtained by representative sampling from herds and animals in Bangladesh are intended and suggested.

CONCLUSIONS
The occurrence of C. fetus in preputial smegma samples of breeding bulls indicates that bulls can act as a potential reservoirs for the transmission of this pathogen to healthy female cattle in Bangladesh. Therefore, annual screening of bulls for evaluation of breeding soundness should be mandatory. The coverage of artificial insemination with C.
fetus infection free bulls' semen can be broaden to minimise reproductive failures and support to the livelihood of marginal dairy farmers.
Moreover, the large-scale occurrence of multidrug-resistant strains of C. fetus in bull samples in this study highlights the clear need to implement the judicial use of antimicrobial agents in veterinary treatment to protect for both animal and human health. Finally, the results of this study indicate the critical need to develop and implement official guidelines related to breeding bull screening, culling and segregation of infected animals to reduce C. fetus infection in bull herds and support to livestock production and food security in Bangladesh.
tion of bull farms under this study. The authors are thankful to farmers, farm managers/attendants for their cooperation during field survey data collection and subsequent animal sampling.

CONFLICT OF INTEREST
The authors declare that they have no competing interest exists regarding financial or personal that could have appeared to influence the work reported in this paper.

ETHICAL APPROVAL AND INFORMED CONSENTS
The Animal Welfare and Experimentation Ethical Committee (AWEEC) of Bangladesh Agricultural University approved the study under the sanction no. AWEEC/BAU/2019(45). A verbal permission was taken from the owner/manager of the cattle farms during data collection and subsequent animal sampling. Animals were handled humanely to lessen suffering.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are included in the manuscript.

PEER REVIEW
The peer review history for this article is available at https://publons. com/publon/10.1002/vms3.831